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BioRxiv : the Preprint Server For... Jun 2024Under stress conditions, cells reprogram their molecular machineries to mitigate damage and promote survival. Ubiquitin signaling is globally increased during oxidative...
Under stress conditions, cells reprogram their molecular machineries to mitigate damage and promote survival. Ubiquitin signaling is globally increased during oxidative stress, controlling protein fate and supporting stress defenses at several subcellular compartments. However, the rules driving subcellular ubiquitin localization to promote these concerted response mechanisms remain understudied. Here, we show that K63-linked ubiquitin chains, known to promote proteasome-independent pathways, accumulate primarily in non-cytosolic compartments during oxidative stress induced by sodium arsenite in mammalian cells. Our subcellular ubiquitin proteomic analyses of non-cytosolic compartments expanded 10-fold the pool of proteins known to be ubiquitinated during arsenite stress (2,046) and revealed their involvement in pathways related to immune signaling and translation control. Moreover, subcellular proteome analyses revealed proteins that are recruited to non-cytosolic compartments under stress, including a significant enrichment of helper ubiquitin-binding adaptors of the ATPase VCP that processes ubiquitinated substrates for downstream signaling. We further show that VCP recruitment to non-cytosolic compartments under arsenite stress occurs in a ubiquitin-dependent manner mediated by its adaptor NPLOC4. Additionally, we show that VCP and NPLOC4 activities are critical to sustain low levels of non-cytosolic K63-linked ubiquitin chains, supporting a cyclical model of ubiquitin conjugation and removal that is disrupted by cellular exposure to reactive oxygen species. This work deepens our understanding of the role of localized ubiquitin and VCP signaling in the basic mechanisms of stress response and highlights new pathways and molecular players that are essential to reshape the composition and function of the human subcellular proteome under dynamic environments.
PubMed: 38948861
DOI: 10.1101/2024.06.20.598218 -
Biomeditsinskaia Khimiia Jun 2024Renalase (RNLS) is a recently discovered protein that plays an important role in the regulation of blood pressure by acting inside and outside cells. Intracellular RNLS...
Renalase (RNLS) is a recently discovered protein that plays an important role in the regulation of blood pressure by acting inside and outside cells. Intracellular RNLS is a FAD-dependent oxidoreductase that oxidizes isomeric forms of β-NAD(P)H. Extracellular renalase lacking its N-terminal peptide and cofactor FAD exerts various protective effects via non-catalytic mechanisms. Certain experimental evidence exists in the literature that the RP220 peptide (a 20-mer peptide corresponding to the amino acid sequence RNLS 220-239) reproduces a number of non-catalytic effects of this protein, acting on receptor proteins of the plasma membrane. The possibility of interaction of this peptide with intracellular proteins has not been studied. Taking into consideration the known role of RNLS as a possible antihypertensive factor, the aim of this study was to perform proteomic profiling of the kidneys of normotensive and hypertensive rats using RP220 as an affinity ligand. Proteomic (semi-quantitative) identification revealed changes in the relative content of about 200 individual proteins in the kidneys of hypertensive rats bound to the affinity sorbent as compared to the kidneys of normotensive animals. Increased binding of SHR renal proteins to RP220 over the normotensive control was found for proteins involved in the development of cardiovascular pathology. Decreased binding of the kidney proteins from hypertensive animals to RP220 was noted for components of the ubiquitin-proteasome system, ribosomes, and cytoskeleton.
Topics: Animals; Rats; Kidney; Hypertension; Rats, Inbred SHR; Proteomics; Monoamine Oxidase; Male; Ligands; Peptides; Proteome
PubMed: 38940203
DOI: 10.18097/PBMC20247003145 -
Frontiers in Immunology 2024TRIM21 is a pivotal effector in the immune system, orchestrating antibody-mediated responses and modulating immune signaling. In this comprehensive study, we focus on...
TRIM21 is a pivotal effector in the immune system, orchestrating antibody-mediated responses and modulating immune signaling. In this comprehensive study, we focus on the interaction of TRIM21 with Fc engineered antibodies and subsequent implications for viral neutralization. Through a series of analytical techniques, including biosensor assays, mass photometry, and electron microscopy, along with structure predictions, we unravel the intricate mechanisms governing the interplay between TRIM21 and antibodies. Our investigations reveal that the TRIM21 capacity to recognize, bind, and facilitate the proteasomal degradation of antibody-coated viruses is critically dependent on the affinity and avidity interplay of its interactions with antibody Fc regions. We suggest a novel binding mechanism, where TRIM21 binding to one Fc site results in the detachment of PRYSPRY from the coiled-coil domain, enhancing mobility due to its flexible linker, thereby facilitating the engagement of the second site, resulting in avidity due to bivalent engagement. These findings shed light on the dual role of TRIM21 in antiviral immunity, both in recognizing and directing viruses for intracellular degradation, and demonstrate its potential for therapeutic exploitation. The study advances our understanding of intracellular immune responses and opens new avenues for the development of antiviral strategies and innovation in tailored effector functions designed to leverage TRIM21s unique binding mode.
Topics: Humans; Ribonucleoproteins; Protein Binding; Antibodies, Neutralizing; Immunoglobulin Fc Fragments; Protein Engineering; Antibodies, Viral; Antibody Affinity; Animals
PubMed: 38938560
DOI: 10.3389/fimmu.2024.1401471 -
Journal of Plant Physiology Jun 2024The F-box protein (FBP) family plays diverse functions in the plant kingdom, with the function of many members still unrevealed. In this study, a specific FBP called...
The F-box protein (FBP) family plays diverse functions in the plant kingdom, with the function of many members still unrevealed. In this study, a specific FBP called PmFBK2, containing Kelch repeats from Persicaria minor, was functionally investigated. Employing the yeast two-hybrid (Y2H) assay, PmFBK2 was found to interact with Skp1-like proteins from P. minor, suggesting its potential to form an E3 ubiquitin ligase, known as the SCF complex. Y2H and co-immunoprecipitation tests revealed that PmFBK2 interacts with full-length PmGID1b. The interaction marks the first documented binding between these two protein types, which have never been reported in other plants before, and they exhibited a negative effect on gibberellin (GA) signal transduction. The overexpression of PmFBK2 in the kmd3 mutant, a homolog from Arabidopsis, demonstrated the ability of PmFBK2 to restore the function of the mutated KMD3 gene. The function restoration was supported by morphophysiological and gene expression analyses, which exhibited patterns similar to the wild type (WT) compared to the kmd3 mutant. Interestingly, the overexpression of PmFBK2 or PmGID1b in Arabidopsis had opposite effects on rosette diameter, seed weight, and plant height. This study provides new insights into the complex GA signalling. It highlights the crucial roles of the interaction between FBP and the GA receptor (GID1b) in regulating GA responses. These findings have implications for developing strategies to enhance plant growth and yield by modulating GA signalling in crops.
PubMed: 38936241
DOI: 10.1016/j.jplph.2024.154299 -
International Journal of Molecular... Jun 2024The SPRY domain-containing SOCS box proteins SPSB1, SPSB2, and SPSB4 utilize their SPRY/B30.2 domain to interact with a short region in the N-terminus of inducible...
The SPRY domain-containing SOCS box proteins SPSB1, SPSB2, and SPSB4 utilize their SPRY/B30.2 domain to interact with a short region in the N-terminus of inducible nitric oxide synthase (iNOS), and recruit an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in the proteasomal degradation of iNOS. Inhibitors that can disrupt the endogenous SPSB-iNOS interactions could be used to augment cellular NO production, and may have antimicrobial and anticancer activities. We previously reported the rational design of a cyclic peptide inhibitor, cR8, cyclo(RGDINNNV), which bound to SPSB2 with moderate affinity. We, therefore, sought to develop SPSB inhibitors with higher affinity. Here, we show that cyclic peptides cR7, cyclo(RGDINNN), and cR9, cyclo(RGDINNNVE), have ~6.5-fold and ~2-fold, respectively, higher SPSB2-bindng affinities than cR8. We determined high-resolution crystal structures of the SPSB2-cR7 and SPSB2-cR9 complexes, which enabled a good understanding of the structure-activity relationships for these cyclic peptide inhibitors. Moreover, we show that these cyclic peptides displace full-length iNOS from SPSB2, SPSB1, and SPSB4, and that their inhibitory potencies correlate well with their SPSB2-binding affinities. The strongest inhibition was observed for cR7 against all three iNOS-binding SPSB proteins.
Topics: Peptides, Cyclic; Humans; Suppressor of Cytokine Signaling Proteins; Nitric Oxide Synthase Type II; Oligopeptides; Protein Binding; Structure-Activity Relationship
PubMed: 38928469
DOI: 10.3390/ijms25126764 -
Zhongguo Shi Yan Xue Ye Xue Za Zhi Jun 2024Multiple myeloma (MM) is an incurable malignant plasma cell diseases, the incidence of which is increasing year by year. The application of immunomodulators drugs,... (Review)
Review
Multiple myeloma (MM) is an incurable malignant plasma cell diseases, the incidence of which is increasing year by year. The application of immunomodulators drugs, proteasome inhibitors, anti-CD38 antibodies, CAR-T, and HSCT have significantly improved the prognosis of patients with MM, however new therapeutic tools need to be developed to improve the prognosis of patients with relapsed/refractory after conventional regimens treatment. Bispecific antibodies are a novel immunotherapeutic approach that generates immune synapses by binding to targets on malignant plasma cells and cytotoxic immune effector cells (T cells/natural killer cells), leading to T/NK cells activation and malignant plasma cell lysis. Several preclinical and phase I clinical studies have shown good efficacy, bringing new possibilities for patients with relapsed/refractory MM to improve their prognosis in the future in combination with the rest of the treatment options. This article summarizes the classification of bispecific antibodies developed in recent years, and the results of preclinical and clinical trials, which will provide some reference for treating MM.
Topics: Humans; Antibodies, Bispecific; Multiple Myeloma; Immunotherapy; Killer Cells, Natural; Prognosis; T-Lymphocytes
PubMed: 38926994
DOI: 10.19746/j.cnki.issn.1009-2137.2024.03.046 -
Scientific Reports Jun 2024Excess amounts of histones in the cell induce mitotic chromosome loss and genomic instability, and are therefore detrimental to cell survival. In yeast, excess histones...
Excess amounts of histones in the cell induce mitotic chromosome loss and genomic instability, and are therefore detrimental to cell survival. In yeast, excess histones are degraded by the proteasome mediated via the DNA damage response factor Rad53. Histone expression, therefore, is tightly regulated at the protein level. Our understanding of the transcriptional regulation of histone genes is far from complete. In this study, we found that calcineurin inhibitor treatment increased histone protein levels, and that the transcription factor NFATc1 (nuclear factor of activated T cells 1) repressed histone transcription and acts downstream of the calcineurin. We further revealed that NFATc1 binds to the promoter regions of many histone genes and that histone transcription is downregulated in a manner dependent on intracellular calcium levels. Indeed, overexpression of histone H3 markedly inhibited cell proliferation. Taken together, these findings suggest that NFATc1 prevents the detrimental effects of histone H3 accumulation by inhibiting expression of histone at the transcriptional level.
Topics: NFATC Transcription Factors; Histones; Calcineurin; Humans; Cell Proliferation; Gene Expression Regulation; Promoter Regions, Genetic; Signal Transduction; Transcription, Genetic; Calcium
PubMed: 38926604
DOI: 10.1038/s41598-024-65769-9 -
Nature Communications Jun 2024Targeted protein degradation (TPD) relies on small molecules to recruit proteins to E3 ligases to induce their ubiquitylation and degradation by the proteasome. Only a...
Targeted protein degradation (TPD) relies on small molecules to recruit proteins to E3 ligases to induce their ubiquitylation and degradation by the proteasome. Only a few of the approximately 600 human E3 ligases are currently amenable to this strategy. This limits the actionable target space and clinical opportunities and thus establishes the necessity to expand to additional ligases. Here we identify and characterize SP3N, a specific degrader of the prolyl isomerase FKBP12. SP3N features a minimal design, where a known FKBP12 ligand is appended with a flexible alkylamine tail that conveys degradation properties. We found that SP3N is a precursor and that the alkylamine is metabolized to an active aldehyde species that recruits the SCF ligase for FKBP12 degradation. Target engagement occurs via covalent adduction of Cys326 in the FBXO22 C-terminal domain, which is critical for ternary complex formation, ubiquitylation and degradation. This mechanism is conserved for two recently reported alkylamine-based degraders of NSD2 and XIAP, thus establishing alkylamine tethering and covalent hijacking of FBXO22 as a generalizable TPD strategy.
Topics: Humans; Proteolysis; Ubiquitination; F-Box Proteins; HEK293 Cells; Tacrolimus Binding Protein 1A; Ubiquitin-Protein Ligases; Amines; Proteasome Endopeptidase Complex; Ligands; Receptors, Cytoplasmic and Nuclear
PubMed: 38926334
DOI: 10.1038/s41467-024-49739-3 -
Science Translational Medicine Jun 2024Targeting ferroptosis for cancer therapy has slowed because of an incomplete understanding of ferroptosis mechanisms under specific pathological contexts such as...
Targeting ferroptosis for cancer therapy has slowed because of an incomplete understanding of ferroptosis mechanisms under specific pathological contexts such as tumorigenesis and cancer treatment. Here, we identify TRPML1-mediated lysosomal exocytosis as a potential anti-ferroptotic process through genome-wide CRISPR-Cas9 activation and kinase inhibitor library screening. AKT directly phosphorylated TRPML1 at Ser and inhibited K552 ubiquitination and proteasome degradation of TRPML1, thereby promoting TRPML1 binding to ARL8B to trigger lysosomal exocytosis. This boosted ferroptosis defense of AKT-hyperactivated cancer cells by reducing intracellular ferrous iron and enhancing membrane repair. Correlation analysis and functional analysis revealed that TRPML1-mediated ferroptosis resistance is a previously unrecognized feature of AKT-hyperactivated cancers and is necessary for AKT-driven tumorigenesis and cancer therapeutic resistance. TRPML1 inactivation or blockade of the interaction between TRPML1 and ARL8B inhibited AKT-driven tumorigenesis and cancer therapeutic resistance in vitro and in vivo by promoting ferroptosis. A synthetic peptide targeting TRPML1 inhibited AKT-driven tumorigenesis and enhanced the sensitivity of AKT-hyperactivated tumors to ferroptosis inducers, radiotherapy, and immunotherapy by boosting ferroptosis in vivo. Together, our findings identified TRPML1 as a therapeutic target in AKT-hyperactivated cancer.
Topics: Ferroptosis; Humans; Proto-Oncogene Proteins c-akt; Animals; Cell Line, Tumor; Neoplasms; Phosphorylation; Lysosomes; Mice; Carcinogenesis; Ubiquitination; ADP-Ribosylation Factors
PubMed: 38924427
DOI: 10.1126/scitranslmed.adk0330 -
The Plant Cell Jun 2024Abscisic acid (ABA) signaling is crucial for plant responses to various abiotic stresses. The Arabidopsis (Arabidopsis thaliana) transcription factor ABA INSENSITIVE 5...
Abscisic acid (ABA) signaling is crucial for plant responses to various abiotic stresses. The Arabidopsis (Arabidopsis thaliana) transcription factor ABA INSENSITIVE 5 (ABI5) is a central regulator of ABA signaling. ABI5 BINDING PROTEIN 1 (AFP1) interacts with ABI5 and facilitates its 26S-proteasome-mediated degradation, although the detailed mechanism has remained unclear. Here, we report that an ABA-responsive U-box E3 ubiquitin ligase, PLANT U-BOX 35 (PUB35), physically interacts with AFP1 and ABI5. PUB35 directly ubiquitinated ABI5 in a bacterially reconstituted ubiquitination system and promoted ABI5 protein degradation in vivo. ABI5 degradation was enhanced by AFP1 in response to ABA treatment. Phosphorylation of the T201 and T206 residues in ABI5 disrupted the ABI5-AFP1 interaction and affected the ABI5-PUB35 interaction and PUB35-mediated degradation of ABI5 in vivo. Genetic analysis of seed germination and seedling growth showed that pub35 mutants were hypersensitive to ABA as well as to salinity and osmotic stresses, whereas PUB35 overexpression lines were hyposensitive. Moreover, abi5 was epistatic to pub35, whereas the pub35-2 afp1-1 double mutant showed a similar ABA response to the two single mutants. Together, our results reveal a PUB35-AFP1 module involved in fine-tuning ABA signaling through ubiquitination and 26S-proteasome-mediated degradation of ABI5 during seed germination and seedling growth.
PubMed: 38924024
DOI: 10.1093/plcell/koae194